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Multiplex Immunoassays in the Development of Vaccines Against Enteric Pathogens



ACQUISITION PROGRAM: Office of the Principal Assistant for Acquisition

OBJECTIVE: Develop an efficient, cost-effective serum-based multiplex assay platform that will identify vaccine candidates, determine immune responses and serve as a potent diagnostic tool for epidemiological and clinical studies.

DESCRIPTION: Based on their impact on the military and global health, the United States Department of Defense (DoD) has placed a high priority on the development of vaccines against enteric pathogens causing diarrhea. Enterotoxigenic Escherichia coli (ETEC), Shigella, Campylobacter and norovirus are significant causes of diarrhea and represent significant targets of military, industry, academic and non-governmental (PATH Global Health) vaccine programs.  These pathogens cause a high level of morbidity and significantly impact the military in lost manpower days, reduced effectiveness and increased treatment costs.  The emerging awareness of post-infection complications further adds to the impact of these infections.  In addition, these same pathogens are associated with hundreds of thousands of deaths among children under 5 in the developing world.  Specific knowledge regarding the potential risks for infectious diseases in certain areas is critical to preventing disease as well as directing vaccine research efforts. Key areas in the development of successful vaccines are the identification of novel antigen targets, determination of the immunogenicity of a candidate vaccine, development of efficient means to determine prior pathogen exposure, and identification of correlates of protective immunity.

At present, the gold standard immunoassay for detecting prior exposures (non-stool-based) to infectious disease agents is the enzyme linked immunosorbent assay (ELISA).  In this relatively laborious assay, antibodies in serum samples are tested for binding to antigens specific for a given enteric pathogen.  Such assays have significant limitations with respect to the amount of sample required, technical man-hours (6-24 hours) to complete the assay, and an inability to multiplex in a single well, the latter resulting in increased costs and reagents. Thus, a more efficient assay is desired. 

An ideal immunoassay would allow for the assaying of sera from exposed individuals against a panel of pathogen components that would provide valuable information regarding the response to a pathogen-specific vaccine, and the risk for a given infectious disease exposure and associated chronic health outcomes within a population subset.  In addition, the flexibility of the platform would allow for customizing the assay for a specific disease agent, which would aid in vaccine discovery, as well as, for testing of immune responses against candidate vaccines undergoing clinical trials.  Lastly, the ideal assay system would not be cost-prohibitive and allow for dissemination to and standardization in multiple research sites.

PHASE I: This phase will demonstrate the feasibility of the immunoassay platform to detect exposure(s) of individuals to ETEC, Shigella, Campylobacter and norovirus utilizing defined historical serum samples archived by NMRC and/or samples from the DoD serum repository. The use of human samples with a known medical history of the targeted infection will allow for determination of the sensitivity and accuracy of the assay in detecting responses induced by natural infection.  In addition, this Phase will also evaluate the flexibility of the platform to allow for customizable assays specific to a single pathogen or vaccine that will be tested using existing animal models.  Results will be compared using current methodologies (e.g. ELISA) to determine the reproducibility and repeatability of the multiplex assay.

PHASE II: In this Phase, the immunoassay developed in Phase I will be validated using human serum samples obtained through an ongoing multisite clinical research study (TravMil) based at DoD travel medicine clinics investigating the epidemiology of travel and deployment related infectious disease threats to U.S Department of Defense (DoD) active duty members and beneficiaries. The organism-specific relative risk of traveler’s diarrhea among DoD travelers as determined by pre- and post-travel serological testing and PCR amplification of organism-specific genetic material collected by participants during illness on a stool filter paper card. This phase will further demonstrate the ability of the assay platform to identify and differentiate between exposures to enteric pathogens.  Furthermore, this Phase will provide a practical implementation of the assay for use in the clinic and/or field.

PHASE III: The developed immunoassay would be a significant aid to global health initiatives focused on preventing enteric diseases due to these pathogens.  Military, industry, academic and public health institutions would benefit greatly from the described assay in aspects of vaccine research and epidemiological measures.  The end-state of this technology would allow for the rapid identification of immune responses to various components of an infectious agent, which would be invaluable in discovering new vaccine candidates and defining exposures to enteric infections of interest.  In addition, this assay will allow for using reduced amounts of valuable samples (e.g. clinical specimens) while also significantly increasing throughput while simultaneously reducing work time.  Spin-off technologies would include customizing the assay to detect additional infectious agents that have relevance not only to human health, but also veterinary health and/or the food industry. 

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